EPO Merck Compound

The recombinant EPO-binding protein (rEBP) is the ECD of the EPOR that has been produced in E. coli, refolded, and characterized for binding activity. It has been used extensively to screen peptide libraries in search of a peptide agonist of EPOR. Using HTS of a chemical library and rEBP, scientists at Merck Research Laboratories identified a small-molecule EPOR antagonist, a biphenyl indole, which they were able to oligomerize and convert into an EPOR agonist (37). The antagonist inhibited binding of 125I-EPO to rEBP with a median inhibitory concentration (IC50) of 60 iM. An EPOR agonist was synthesized by attaching eight copies of the small molecule antagonist to a polyamidoamino-octa-4-hydroxymethylbenzamide support using a chemical linker. This compound inhibited binding of 125I-EPO to the rEBP with a IC50 of 4.4 |M in the EPOR binding assay. Although both compounds were tested in an EPOR dimerization assay, only the small-molecule agonist induced dimerization of soluble EPOR.

The results from further experimentation showed that the biphenyl indole agonist could indeed function as a successful EPO mimetic (37). Screening the compound against a cell line that expresses a functional human EPOR using a luciferase protein reporter gene driven by activated STAT transcription factors indicated that this agonist induced cellular transcription (Fig. 2) and proliferation in cells expressing EPOR. Furthermore, the Merck compound supported the proliferation of several tumor cell lines

Fig. 2. Induction of luciferase activity in BAF3/LUC/EPOR cells expressing human EPOR. BAF3/LUC/EPOR cells containing a stably integrated luciferase gene under the control of a STAT-binding element were treated with varying amounts of erythropoietin (EPO) or small molecule for 16 h. The luciferase activity is expressed in relative light units (RLU). Data are the mean (± SEM) of two to three independent experiments performed in triplicate. The x-axis reflects the concentration of EPO (closed circles) or small molecule (closed squares) in unit/mL or |iM, respectively. Assays were performed in 1% dimethyl sulfoxide. (Adapted with permission from ref. 37. Copyright 1999 National Academy of Sciences USA.)

Fig. 2. Induction of luciferase activity in BAF3/LUC/EPOR cells expressing human EPOR. BAF3/LUC/EPOR cells containing a stably integrated luciferase gene under the control of a STAT-binding element were treated with varying amounts of erythropoietin (EPO) or small molecule for 16 h. The luciferase activity is expressed in relative light units (RLU). Data are the mean (± SEM) of two to three independent experiments performed in triplicate. The x-axis reflects the concentration of EPO (closed circles) or small molecule (closed squares) in unit/mL or |iM, respectively. Assays were performed in 1% dimethyl sulfoxide. (Adapted with permission from ref. 37. Copyright 1999 National Academy of Sciences USA.)

that express human or mouse EPOR and induced differentiation of human progenitor cells into cells of erythrocytic lineage. Although recombinant human EPO (rHuEPO) was much more potent than the Merck agonist in all these experiments, an important proof of principle was obtained.